Rational design of an estrogen receptor mutant with altered DNA-binding specificity

Abstract

Although artificial C2-H2 zinc fingers can be designed to recognize specific DNA sequences, it remains unclear to which extent nuclear receptor C4 zinc fingers can be tailored to bind novel DNA elements. Steroid receptors bind as dimers to palindromic response elements differing in the two central base pairs of repeated motifs. Predictions based on one amino acid—one base-pair relationships may not apply to estrogen receptors (ERs), which recognize the two central base pairs of estrogen response elements (EREs) via two charged amino acids, each contacting two bases on opposite DNA strands. Mutagenesis of these residues, E203 and K210 in ERα, indicated that both contribute to ERE binding. Removal of the electric charge and steric constraints associated with K210 was required for full loss of parental DNA-binding specificity and recognition of novel sequences by E203 mutants. Although some of the new binding profiles did not match predictions, the double mutation E203R-K210A generated as predicted a mutant ER that was transcriptionally active on palindromes of PuGCTCA motifs, but not on consensus EREs. This study demonstrates the feasibility of designing C4 zinc finger mutants with novel DNA-binding specificity, but also uncovers limitations of this approach.

Genomic Responses from the Estrogen-responsive Element-dependent Signaling Pathway Mediated by Estrogen Receptor α Are Required to Elicit Cellular Alterations

“…It appears that the nature of charge and steric constraint of residues in the DNA recognition helix of ER␣ contributes to the recognition of and the extent of binding to ERE (38). Based on these findings and our preliminary observations, we introduced single or multiple amino acid changes in the DNA recognition helix of ER␣ 203/4 to generate an ER variant that lacks the ability to interact with ERE.…”

“…Recent structural studies uncovered a further complexity in the binding of the human ER␣ to an ERE (38). It appears that the nature of charge and steric constraint of residues in the DNA recognition helix of ER␣ contributes to the recognition of and the extent of binding to ERE (38).…”

“…Based on these findings and our preliminary observations, we introduced single or multiple amino acid changes in the DNA recognition helix of ER␣ 203/4 to generate an ER variant that lacks the ability to interact with ERE. Of mutant receptors, the replacement of positively charged Arg 211 , which is a conserved residue among the nuclear hormone receptors and is critical for the interaction with bases in a cognate response element (35,38), with negatively charged Glu residue in ER␣ 203/4 was sufficient to generate an ER␣ mutant (ER␣ 203/4/11 ) (Fig. 1A) functional only at the ERE-independent signaling pathway ( Figs.…”

Transcriptional modulation of BCRP gene to reverse multidrug resistance by toremifene in breast adenocarcinoma cells

“…The consensus ERE consisting of palindromes of PuGGTCA motifs separated by a three base-pair spacer is bound by ERs with high affinity, with one receptor interacting with each PuGGTCA motif [18][19][20][21]. ER typically functions as a transcription factor to regulate specific gene expression by interacting specifically with an estrogen-responsive element of the enhancer region [22].…”

Utilização de informações termodinâmicas e estruturais na predição de sítios de ligação de receptores nucleares ao DNA: uma abordagem computacional

“…Os nucleotídeos dos P-box estão sublinhados. Imagem adaptada de (Nguyen et al, 2007 É importante observar que, dados dois monômeros, existem duas maneiras de ligálos a uma fita de DNA. A primeira delas é fazê-lo simetricamente, como é o caso do ER-(Figura 1.5).…”